Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas

The Pacific oyster, Crassostrea gigas, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in C. gigas, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the C. gigas autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of C. gigas. This study allows the characterization of the complete C. gigas autophagy pathway for the first time.
Abbreviations: ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.

太平洋牡蛎（Pacific oyster，学名*Crassostrea gigas*）是一种兼具重要商业食用价值的软体双壳类动物。其在养殖过程中会遭受环境胁迫并罹患多种病害。自噬通路（autophagy pathway）参与诸多细胞生理过程，包括饥饿响应、细胞死亡及微生物清除等。长牡蛎中同样存在自噬现象，且在抗感染免疫应答中发挥关键作用。尽管该过程在多数动物中已有充分研究且序列高度保守，但在软体动物中的相关机制仍不甚明晰。截至目前，尚无研究对软体双壳类动物的自噬分子机制进行全面系统的阐述。
本研究以人类与酵母的ATG（autophagy related，自噬相关）蛋白序列为参照，结合公共数据库Uniprot（通用蛋白质资源库，Universal Protein Resource）与NCBI（美国国家生物技术信息中心，National Center for Biotechnology Information），对长牡蛎自噬通路的蛋白成员进行鉴定。最终在太平洋牡蛎中共鉴定得到35个自噬相关蛋白。研究人员针对部分基因开展了RACE-PCR（cDNA末端快速扩增-聚合酶链式反应，rapid amplification of cDNA-ends by polymerase chain reaction）实验，并采用分子生物学（实时荧光定量PCR，real-time PCR）与蛋白质检测技术（蛋白质印迹、免疫组织化学（immunohistochemistry，IHC）），对太平洋牡蛎不同组织中ATG12、ATG9、BECN1、MAP1LC3、MTOR及SQSTM1的表达与定位情况进行了分析。与人类及酵母的同源蛋白比对结果显示，长牡蛎自噬通路与人类的同源性较高。研究结果同时证实，关键自噬基因及其编码蛋白在长牡蛎所有被检测的组织中均有表达。本研究首次完成了长牡蛎完整自噬通路的系统鉴定与特征分析。

缩写说明：
ATG：自噬相关（autophagy related）
Atg1/ULK：UNC-51样自噬激活激酶（unc-51 like autophagy activating kinase）
ATG7：自噬相关蛋白7（autophagy related 7）
ATG9：自噬相关蛋白9（autophagy related 9）
ATG12：自噬相关蛋白12（autophagy related 12）
BECN1：Beclin 1
BSA：牛血清白蛋白（bovine serum albumin）
cDNA：互补脱氧核糖核酸（complementary deoxyribonucleic acid）
DNA：脱氧核糖核酸（deoxyribonucleic acid）
GABARAP：GABA_A型受体相关蛋白（GABA type A receptor-associated protein）
IHC：免疫组织化学（immunohistochemistry）
MAP1LC3/LC3/Atg8：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）
MTOR：雷帕霉素靶蛋白激酶（mechanistic target of rapamycin kinase）
NCBI：美国国家生物技术信息中心（National Center for Biotechnology Information）
ORF：开放阅读框（open reading frame）
PBS：磷酸盐缓冲液（phosphate-buffered saline）
PCR：聚合酶链式反应（polymerase chain reaction）
PtdIns3K：Ⅲ型磷脂酰肌醇3-激酶（class III phosphatidylinositol 3-kinase）
RACE-PCR：cDNA末端快速扩增-聚合酶链式反应（rapid amplification of cDNA-ends by polymerase chain reaction）
RNA：核糖核酸（ribonucleic acid）
SQSTM1：自噬底物结合蛋白1（sequestosome 1）
Uniprot：通用蛋白质资源库（Universal Protein Resource）
WIPI：WD重复结构域磷酸肌醇互作蛋白（WD repeat domain, phosphoinositide interacting）